1. Elucidation of the involvement of the second extracellular loop (ECL2) and transmembrane residues of CCR5 in inhibitor binding and HIV-1 fusion.We examined the interactions between CCR5 and novel CCR5 inhibitors containing the SDP scaffold, AK530 and AK317, both of which were docked into the hydrophobic cavity located between the upper transmembrane domain and ECL2 of CCR5 (67). Figure 11 shows the lipophilic potential mapped on the binding cavity of CCR5 and relative binding modes of AK530 and AK317. Molecular dynamics simulations for inhibitor-unbound CCR5 showed hydrogen bond interactions among transmembrane residues Y108, E283, and Y251, which were crucial for HIV-1-gp120/sCD4 complex binding and HIV-1 fusion. The data should not only help delineate the dynamics of CCR5 following inhibitor binding but also aid in designing CCR5 inhibitors that are more potent against HIV-1 and prevent or delay the emergence of resistant HIV-1 variants. We continued the design, synthesis, and evaluation of different CCR5 inhibitors in collaboration with Professor Ghosh of Purdue University. We initially started from a published CCR5 antagonist from the literature, and built structural models of its interaction with CCR5. Over the last four years, the design, synthesis and biological evaluation of more than 100 inhibitors has been carried out and several novel and potent inhibitors of CCR5 have been discovered in our study. Identified CCR5 antagonists include GRL-117, GRL-10007, and GRL-10018, which proved to be active against R5-HIV-1 with antiretroviral IC50 values of 0.6 nM, 1.4 nM and 2.9 nM, respectively. The IC50 value of MVC, the only approved CCR5 inhibitor at present, was 0.7 nM in the same assay. These newly identified compounds were also potent against vicriviroc (VCV)-resistant R5-HIV-1. The IC50 value of GRL-10007 decreased by 5.3-fold against VCV-resistant virus whereas the activity of MVC decreased by 10.6-fold. Experiments using CCR5 antibody revealed that these compounds were bound to ECL2 of CCR5 as other reported CCR5 antagonists do. These compounds also effectively blocked the interactions between CC-chemokines and CCR5, but GRL-10007 only partially blocked RANTES binding to CCR5 and RANTES-induced Ca2+ flux (Nakata, Das, Ghosh and Mitsuya: manuscript in preparation). Of note, the RANTES binding and Ca2+ flux profile of GRL-10007 were similar to those of APL, suggesting that, as was in the case of APL, GRL-10007 may have highly specific binding to CCR5 without overly inhibiting CCR5-chemokine interactions. 2. Determination of binding mode of the new CCR5 inhibitors and elucidation of structure-activity relationshipsWe built structural models of newly identified CCR5 inhibitors to gain insight to the mechanism of potency of such inhibitors. A model of the interaction of GRL117 is shown in Figure 13. GRL117 binds in a cavity formed within the transmembrane helices and ECL2. The models suggest that GRL117 has polar interactions with Y37, C178, K191, and T195. Y37 is located in transmembrane-1 (TM-1), K191 and T195 are located in TM-5. C178 is located in ECL2 and is highly conserved amongst class-A GPCRs. These residues are also important for gp120 fusion, and for the binding of APL and other CCR5 inhibitors.&#12288;We postulated that interactions of inhibitors with CCR5 residues that are important for gp120 fusion may cause conformation change in CCR5, and may represent a salient molecular mechanism enabling allosteric inhibition. The interactions of GRL117 with such residues might be responsible for its potent antiretroviral IC50 of 0.6 nM. 3. Study of impacts of amino acid substitutions on CD4/gp120-CXCR4-induced fusion and identification of lead CXCR4 inhibitors.We first attempted to examine the impacts of amino acid substitution on CD4/gp120-CXCR4-induced fusion by introducing amino acid substitutions into human wild-type CXCR4 CXCR4WT)and determined the changes in fusion levels, compared to the fusion activity of CXCR4WT (referenced as 100%). We found that amino acid substitutions D97A, D262A, and E288A located in different transmembrane domains resulted in substantial loss of fusion activity. These residues have also been shown to be important for the binding of different CXCR4 inhibitors. As expected, various amino acid substitutions in ECL2, such as A175F, D182A, D187A, R188A and Y190A also resulted in compromised fusion (Figure 14).The availability of the crystal structure of CXCR4 with a small molecule inhibitor greatly aided our efforts to identify lead CXCR4 inhibitors. Out of an initial selection of 16 molecules with virtual screening of 622,000 different molecules, we identified cyclopentane-piperidine analogues as having anti-HIV activity (Das, Maeda, Hayashi, and Mitsuya: manuscript in preparation). Subsequently, other molecules containing the cyclopentane-piperidine scaffold were selected from the general screening library. Such molecules were found to have IC50 values ranging 400 nM to 9 microM in MTT assay. CX-6 and CX-20 were identified as lead molecules, and our data warrants further optimization of the candidates by increasing interactions with other key residues in the active site. We have started collaboration with Professor Ghosh of Purdue University in optimizing the scaffold and the newly identified CXCR4 inhibitors